Voltage gated ion channels are major determinants of membrane excitability. The Kv1.2 potassium channel is expressed widely throughout the brain, but little is known about Kv1.2 regulation in the brain. Using model cell systems, Kv1.2 was found to be regulated by tyrosine and serine/threonine phosphorylation. More recently, we have identified ubiquitylation as being capable of modulating Kv1.2 function. In preliminary studies we show that ubiquitylation affects Kv1.2 endocytosis and recycling and that it does so independently of Kv1.2 degradation. Intriguingly, mutagenesis of ubiquitylation sites within Kv1.2 revealed that distinct patterns of ubiquitylation within Kv1.2 affect channel trafficking in dramatically different ways, some decreasing and some increasing Kv1.2 levels at the cell surface. These preliminary studies employed model cell systems and ectopically expressed wild type or mutant forms of Kv1.2 to reveal a nuanced role for ubiquitylation in Kv1.2 regulation. As informative as it is, however, this approach only suggests how ubiquitylation affects Kv1.2 endogenously expressed in the brain. Given the key role of Kv1.2 in the brain, the complete lack of information on the occurrence and effects of Kv1.2 ubiquitylation there is a striking gap. The current challenge, and goal of this grant proposal, is to determine whether the types of ubiquitylation predicted by our preliminary studies with ectopically expressed Kv1.2 occur in Kv1.2 expressed endogenously within the brain. To do so, we will use AQUA and SILAC mass spectrometry methods to quantitatively determine the amount and intra-molecular patterns of stimulus-induced ubiquitylation of Kv1.2 expressed endogenously within the brain. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms of ion channel regulation in the brain is of tremendous importance to human health. Very little is known about how an important form of protein modification, ubiquitylation, regulates potassium channels expressed endogenously within the brain. The proposed studies address this gap by using mass spectrometry to examine ubiquitin-dependent regulation of the Kv1.2 potassium channel in brain tissue.